Phase plates for cryo-EM Rado Danev Max Planck Institute of - PowerPoint PPT Presentation

Max Planck Institute of Biochemistry Martinsried, Germany MAX PLANCK SOCIETY Phase plates for cryo-EM Rado Danev Max Planck Institute of Biochemistry, Martinsried, Germany. MPI für Biochemie MPI für Biochemie EMBO course 2017, London, UK Max-Planck-Gesellschaft Max Planck Gesellschaft

The Transmission Electron Microscope (TEM) electron source condenser lens(es) object (specimen) plane objective lens back focal (diffraction) plane image forming lens(es) image (observation) plane MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Mathematical model of TEM e  r i ( ) A ( ) r object wave FT e  k Contrast Transfer Function i ( ) (CTF) FT |..| 2 square detection image intensity I r ( ) MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Effect of the phase plate e  r ( ) object wave A ( ) r FT Contrast Transfer Function e    i ( ) i k ( ) k (CTF) + Phase Plate Function FT |..| 2 square detection I r ( ) image intensity MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Phase contrast transfer function – weak object     1 1            2 3 4   CTF ( k ) sin 2 z k C k s     2 4  ( k ) phase shift aberration term MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Ideal case – 90 o phase shift phase plateTEM conventional TEM  k  k sin( ( )) cos( ( )) 300 kV,  =0.001968 nm, C S = 3 mm 300 kV,  =0.001968 nm, C S = 3 mm  z = 93 nm 1.0 1.0  z = 56 nm 0.5 0.5 -sin(  ( k )) cos(  ( k )) 0.0 0.0 -0.5 -0.5  z = 0 nm  z = 0 nm -1.0 -1.0 0 1 2 3 4 5 0 1 2 3 4 5 k [1/nm] k [1/nm] MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Phase plates improve the contrast Conventional cryo-EM VPP cryo-EM 1.5 um defocus in-focus 50 nm MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

TEM imaging modes Conventional Zernike Hilbert diff Conventional TEM Phase Plate TEM (defocus) specimen objective lens back-focal phase plate aperture plane image forming lenses image MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

TEM imaging modes Conventional TEM Zernike Phase Plate Volta Phase Plate CTEM ZPP VPP central central central beam beam beam hole thin film thin film OL aperture MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Volta phase plate - phase shift evolution image series FFT Niquist frequency ~ 4.3A MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

VPP – phase shift vs. beam current Danev et al., “Volta potential phase plate for in - focus phase contrast transmission electron microscopy”, PNAS, 2014 • The phase shift depends on the total dose and not on the dose rate. • MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Bridget’s questions 1. Are phase plates a key to high resolution of small and heterogeneous particles? 2. Can we make them easier to use? 3. What are the remaining issues? 4. Will there be progress in the near future for these devices? MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Bridget’s questions 1. Are phase plates a key to high resolution of small and heterogeneous particles? 2. Can we make them easier to use? 3. What are the remaining issues? 4. Will there be progress in the near future for these devices? MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Theoretical estimates R. Henderson , “The potential and limitations of neutrons, electrons and x -rays for atomic-resolution microscopy of unstained • biological molecules”, Quarterly Reviews of Biophysics , 1995 The smallest particle size for which the orientations can be determined is • approximately 38/ C 2 kDa , where 0 < C ≤ 1 is the contrast relative to that of a perfect phase contrast image. The number of particles required is 38,000/ d , where d is the resolution in Å. • MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Simulated ZPP data – size test Chang et al., “Zernike phase plate cryoelectron microscopy facilitates single particle analysis of unstained asymmetric protein • complexes”, Structure , 2010 500 kDa 290 kDa 100 kDa MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Simulated ZPP data – size test Hall et al. ,”Accurate modeling of single-particle cryo-EM images quantitates the benefits expected from using Zernike phase • contrast”, Journal of Structural Biology , 2011 100 kDa protein MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Simulated ZPP data – heterogeneity test Hall et al. ,”Accurate modeling of single-particle cryo-EM images quantitates the benefits expected from using Zernike phase • contrast”, Journal of Structural Biology , 2011 ~200 kDa protein +- 12.5 kDa +- 33 kDa MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Structures solved with the VPP Calcitonin GPCR 150 kDa, 4.1 Å EMD-8623 Preferred orientation, Smallish size, Smallish size, thick ice low contrast in top views some flexibility GLP-1 GPCR Rpn1 150 kDa, 3.3 Å 100 kDa, 3.8 Å EMD-7039 Smallish size Small size, Small size preferred orientation MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Answer to the 1 st question 1. Are phase plates a key to high resolution of small and heterogeneous particles? • They seem to help < 200 kDa. They may be the key to many < 100 kDa structures. • Heterogeneity is too complex to generally quantify, but phase plates are • expected to improve the performance of classification. • Previous image simulations were done before “the revolution” without taking into account new methods, such as frame alignment and dose weighting. Frame alignment should benefit from the use of a phase plate by using a • finer temporal sampling, i.e. “super fractionation”. Dose weighting improves greatly the performance of the conventional • defocus approach for small particles. • We need up-to-date image simulations! MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Bridget’s questions 1. Are phase plates a key to high resolution of small and heterogeneous particles? 2. Can we make them easier to use? 3. What are the remaining issues? 4. Will there be progress in the near future for these devices? MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

VPP alignments - on-plane condition Volta Phase Plate Volta Phase Plate on-plane off-plane (parallel illumination) (non-parallel illumination) central beam phase plate phase plate back focal plane MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

VPP alignments - beam shift pivot points Correct pivot point setting Incorrect pivot point setting Exposure Exposure Focus Focus specimen specimen plane phase plate back focal plane MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

FEI tools for phase plate navigation Phase Plate Slot • 6 slots x 76 positions  456 fresh areas Single area for ~1 hr operation • Used area of the Phase Plate On Next button MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Answer to the 2 nd question 2. Can we make them easier to use? Using preset imaging states which store all alignments could greatly simplify • their use. An automated phase plate quality test/enumeration could make the VPP • simpler to use by only allowing access to good phase plate positions. Track the usage of each VPP position and go to positions which have had • the longest time to recover? • Future phase plates (laser) may be easier to use in some ways and more difficult in others. MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft

Bridget’s questions 1. Are phase plates a key to high resolution of small and heterogeneous particles? 2. Can we make them easier to use? 3. What are the remaining issues? 4. Will there be progress in the near future for these devices? MPI für Biochemie MPI für Biochemie Max-Planck-Gesellschaft Max Planck Gesellschaft